This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Since early 1990's, heterologous "prime-boost" immunization has been shown to protect against primate lentiviruses in multiple macaque models. However, few studies have compared the efficacy of different prime-boost strategies. In this study, we compared the protective efficacy of DNA, protein, or viral vaccines in different prime-boost combinations. Two cohorts of 24 macaques were primed with recombinant vaccinia or DNA vectors expressing HIV-1 SF162 Env or SIVmac239 Gag/Pol. Animals in each cohort were divided into three groups (N=6/group) and were boosted with DNA, protein;or heterologous viral vector expressing the same antigens. Six control animals in each cohort received empty vectors and adjuvant only. Protective efficacy was evaluated following a high-dose intrarectal challenge of SHIV-SF162P4. After the last immunization, all experimental animals generated SIV- and HIV-specific antibodies. Regardless of the priming, animals boosted with proteins had significantly higher antibody titers, including homologous neutralization antibodies (NtAb). After SHIV-SF162P4 challenge, significant reduction of mean plasma viral load was observed in all immunization groups. An inverse correlation (Spearman's r = -0.819, p lt 0.0001) was observed between NtAb titer on the day of challenge and peak plasma viral load after challenge. Five of 12 animals boosted with proteins showed high NtAb titer and no detectable viremia after challenge, consistent with protection from infection. Although the rest of the immunized animals were all infected, two animals primed with vaccinia and boosted with DNA showed low ( lt 2x10^3 copies/ml) and transient plasma viremia, indicating the potential role of cellular immunity in controlling infection. Together, these results indicate that protective immunity against mucosal infection with a CCR5-using primate lentivirus can be achieved by systemic prime-boost immunization. NtAb played an important role in protection against mucosal infection by SHIVSF162P4. Boosting with protein immunogens appears to be superior to DNA or viral vectors in the generation of antigen-specific antibody responses, including NtAb.